Replica Plating
Replica Plating is generally, a process of stamping a replica of a set of colonies, spots, or streaks from one agar petri dish ("plate") to another, usually from a general growth media onto one or more marker or selective media. In our lab replica plating is used to analyze the results of Tetrad Dissection. Typically the colonies formed by the tetrads on YPD plates are replica plated onto one or more of -Met and -Lys (the markers for the two mating types in our yeast deletion sets), G418 (kanamycin resistance, the deletion cassette used in our deletion collections), and A and alpha tester plates to be re-replica plated into minimal media. This protocol addresses these particular plates; ymmv. If we are running low or are out of these plate you must make some more or ask Justin in the Autoclave room on the first floor. 1. After your dissected tetrads have been in the 30C Incubator for a couple of days, you can replica plate. You'll need Replica Plater, Velvets, selective plates. 2. The replica plater and velvets are in the second from bottom, rightmost drawer under Mark's bench. The selective plates are on the yeast media shelves above the microwave and PCR machines, except for G418 plates, which must be stored at 4C and are in the of the 4C Cold Room, labeled "G418". 3a. Make tester strain lawns for your A and alpha tester plates. This is done by taking 150uL (per YPD plate needed) dH2O in 1.5 ml Eppendorf tubes and twirling in it a toothpick with tester strain yeast on it (the tester strain which is itself alpha, which tests for A, is JO297 and the one which is itself A, which tests for alpha, is JO296; you should have a YPD plate of these two sitting around for just this purpose, and freeze a stock of each the first time you use them- see Freezing Strains). Then use your glass lawn spreader and EtOH in a jar to spread the lawns on YPD plates. Use the burner to flame your ethanoled glass spreader (FIRE!!!), then gently touch a part of the YPD plate with no yeast on it to cool the surface a little before actually contacting the yeast. Spread the lawns and let them dry before moving on to the next step. This is a good time to NOT let a flaming drop of ethanol fall back into your jar of ethanol. If this happens, calmly cover the jar. NOTE::: DO NOT KEEP RESTREAKING YOUR TESTER STRAINS FROM PLATE TO PLATE TO "SAVE" YOU TIME REPULLING THEM. The strains can pick up suppressors, which an entirely nother issue to deal with, so just repull them from the freezer as needed. 3b. Starting with a clean velvet, make a clean unsmeared impression from your ypd plate, then transfer this impression to -lys, -met, G418, any others such as -ura, and finally a tester plate which you spread with A TESTER STRAIN JO296 / AM22? (label this "w/ A=alpha"). 4. Using a second velvet, make a new impression from your YPD plate and transfer to a tester plate which you spread with ALPHA TESTER STRAIN JO297 / AM227 (label this "w/alpha=A"). you may want to end all replica platings with a clean YPD plate as a control to show that cells carry over through all replicas. 5. Place plates, including YPD original, into the 30C Incubator for a couple of days, then photograph, score, and store in the 4C Cold Room if needed. You will then RE-REPLICA your a and alpha tester strain plates to minimal media "B" plates, wait two additional days at 30C, and score for mating types. You may then proceed to freezing down desired strains, and PCR verification of strains by Colony PCR. G418: P/N (company name, item #) 'Replica Plating Tool: P/N: VWR 25395-380 ' 'Velveteen Squares: You can use any VELVETEEN from a fabric store (bring an old velvet for comparison, do not buy fabrics other than VELVETEEN), or use VWR P/N 89033-116 '